Novel non-peptide lead structures forBradykinin B2-receptor antagonists

A series of new non-peptide Bradykinin (BK)B₂-receptor antagonists is reported. These newleads belong to a larger set including bothcommercially or otherwise available potentialcandidates found by proprietary database searchesusing 3D-pharmacophore models as query, and severalbis-benzamidino compounds selected from ourtryptase-like protease inhibitor library on thebasis of topological considerations, derived fromthe same models.Some of these compounds show functional competitiveantagonistic activity, inhibiting {in vitro} the BK-induced contraction of isolated guinea-pig ileum(GPI) and rat uterus with a pA₂ up to 5.3 and7.0, respectively. They display also bindingaffinity (IC₅₀ up to 0.56 M) to the BKB₂-receptor in radioligand binding assays onGPI membrane preparations and on human IMR-90 fetallung fibroblast cells expressing this receptor subtype.Furthermore, the results with the commerciallyavailable compounds, in some cases developed astherapeutics, show that the used 3D-pharmacophoremodel allows to predict to some certainty possibleside actions of potential drugs.     

Formulation of 3D pharmacophore models for bradykinin B2-receptor antagonists

Summary In order to make a further contribution to the elucidation of the essential structural features for bradykinin (BK) antagonism, we extracted 3D pharmacophore models from a training set consisting of nine relatively rigid, small organic, non-peptide molecules, reported to be more or less active, competitive BK B₂-receptor antagonists. This was accomplished by means of the expert system Catalyst^TM. The information contained in one of these models was then used to identify relevant structural features and perform consensus molecular dynamics simulations including, in addition to the non-peptide antagonist set, four prototypical linear and cyclic peptide antagonists, and BK itself. this combined approach allowed us to identify regions of the conformational space shared by this series of compounds, which includes highly flexible, and, hence, otherwise almost inaccessible for conventional conformational sampling techniques, peptide molecules. As a result, we obtained a relatively small number of extended pharmacophore models, whose average, together with the original Catalyst model, could be used to search proprietary 3D databases for potential candidates. The results of such a search are also reported.     

Formulation of 3D pharmacophore models for bradykinin B2-receptor antagonists

In order to make a further contribution to the elucidation of the essential structural features for bradykinin (BK) antagonism, we extracted 3D pharmacophore models from a training set consisting of nine relatively rigid, small organic, non-peptide molecules, reported to be more or less active, competitive BK B₂-receptor antagonists. This was accomplished by means of the expert system Catalyst. The information contained in one of these models was then used to identify relevant structural features and perform consensus molecular dynamics simulations including, in addition to the non-peptide antagonist set, four prototypical linear and cyclic peptide antagonists, and BK itself. This combined approach allowed us to identify regions of the conformational space shared by this series of compounds, which includes highly flexible, and, hence, otherwise almost inaccessible for conventional conformational sampling techniques, peptide molecules. As a result, we obtained a relatively small number of extended pharmacophore models, whose average, together with the original Catalyst model, could be used to search proprietary 3D databases for potential candidates. The results of such a search are also reported.     

Receptor based 3D-QSAR to identify putative binders of Mycobacterium tuberculosis Enoyl acyl carrier protein reductase

In the current study, the applicability and scope of 3D-QSAR models (CoMFA and CoMSIA) to complement virtual screening using 3D pharmacophore and molecular docking is examined and applied to identify potential hits against Mycobacterium tuberculosis Enoyl acyl carrier protein reductase (MtENR). Initially CoMFA and CoMSIA models were developed using series of structurally related arylamides as MtENR inhibitors. Docking studies were employed to position the inhibitors into MtENR active site to derive receptor based 3D-QSAR models. Both CoMFA and CoMSIA yielded significant cross validated q² values of 0.663 and 0.639 and r² values of 0.989 and 0.963, respectively. The statistically significant models were validated by a test set of eight compounds with predictive r² value of 0.882 and 0.875 for CoMFA and CoMSIA. The contour maps from 3D-QSAR models in combination with docked binding structures help to better interpret the structure activity relationship. Integrated with CoMFA and CoMSIA predictive models structure based (3D-pharmacophore and molecular docking) virtual screening have been employed to explore potential hits against MtENR. A representative set of 20 compounds with high predicted IC₅₀ values were sorted out in the present study.     

Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques

Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer’s disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads—Specs1 (IC₅₀ = 3.279 μM) and Spec2 (IC₅₀ = 5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.     

Autoradiographic Localization of [125I-Tyr0]Bradykinin Binding Sites in Brains of Wistar-Kyoto and Spontaneously Hypertensive Rats

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [¹²⁵I-Tyr⁰]bradykinin ([¹²⁵I-Tyr⁰]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [¹²⁵I Tyr⁰]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K _D values in the range of 100 pM and a B _max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg⁹-BK. The B₂ receptor antagonist icatibant inhibited [¹²⁵I-Tyr⁰]BK binding with a K _i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K _i values for the B> ₁ receptor agonist DesArg⁹-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [¹²⁵I-Tyr⁰]BK B₂ binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B₂ receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts

Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E₂ via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca²⁺ kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca²⁺ concentration through the activation of B₂ receptors. Removal of extracellular Ca²⁺ attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca²⁺ pump inhibitor, completely inhibited BK-induced increase of intracellular Ca²⁺. These results suggested that bath-applied BK activated store-operated Ca²⁺ channels (SOCCs) following Ca²⁺ store depletion via B₂ receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl₃, LaCl₃ and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca²⁺ influx via B₂ receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

基于微管蛋白和秋水仙碱结合位点化合物构建3D-药效团新模型及先导物虚拟筛选

基于作用于微管蛋白秋水仙碱结合位点的小分子抑制剂与生物靶标的复合晶体结构,采用分子模拟软件Discovery Studio 3.0的受体-配体药效团产生程序建立了系列3D药效团新模型（M1-M6）,并用20个已知微管抑制剂验证了其可靠性.用新药效团模型对约10000个化合物的数据库进行了虚拟筛选,发现了一些潜在先导物.据此合成的二芳烃胺类新化合物20在抑制人白血细胞K562的初步实验中显示出了明显的细胞形态变化和抑制活性,对多种人癌细胞A549,KB,KBvin和DU145均有较强的抑制活性（GI500.17～1.02μmol/L）,表明以此新构建的药效团模型进行理性设计和寻找新型抗癌先导物的方法具有一定的可行性.     

Allosteric modulation of adenosine receptors

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A₁ receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A₃ allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A₁ and A₃ receptors, whereas limited or no information is available for A_2A and A_2B receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.     

Kinetic Analysis of Estrogen and Antiestrogen Competition for Hypothalamic Cytosol Estrogen Receptors. Evidence for Noncompetitive Ligand-Receptor Interactions

Abstract

The binding of (³H)-estradiol (E₂) to cytoplasmic estrogen receptors isolated from female rat hypothalamus-preoptic area by unlabeled estrogen and antiestrogens (nafoxidine and tamoxifen) was examined when the concentrations of both the agonist (³H)-E₂) and unlabeled competitors were varied over a wide range. Concentrations of unlabeled E₂ up to 10^-10M decreased the affinity of the labeled steroid for its receptor; at higher E₂ concentrations, the apparent number of binding sites (B_max) began to decline. Thus at some concentrations, E₂ may act as a mixed competitive and noncompetitive inhibitor of its own binding to hypothalamic cytosol receptors. The antiestrogens differed markedly from E2 in their interactions with hypothalamic estrogen receptors. Only at relatively high competitor concentrations (e.g., > 10^-9M) did the antagonists appear to competitively inhibit (³H)-E₂-receptor binding. The most striking observation was that tamoxifen and nafoxidine significantly inhibited (³H)-E₂-receptor binding at very low competitor concentrations (e.g., 1 pM), but only slightly inhibited estrogen binding at intermediate concentrations (e.g., 10^-10M). It was proposed that the non-linear, concentration-dependent effects of antiestrogens on the neural estrogen receptors might be due to complex interactions of the antagonists with a non-estrogen binding site.     

Effect of amygdaloid kindling on rat striatal dopamine D1- and D2-receptors

The effect of kindling on dopaminergic (DA) neurotransmission was assessed by measuring dopamine D₁- and D₂-receptor binding in the dorsal and ventral striatum of rats either 2 hours (short-term) or 3–4 weeks (long-term) after the last kindled seizure. Kindling did not have any significant long-term effect on DA D₂-receptor K_d or B_max values in the dorsal or ventral striatum or on DA D₁-receptor parameters in the dorsal striatum. The short-term effect of kindled seizures was to abolish the asymmetry in DA D₂-receptor density observed in the dorsal striatum of control rats. DA D₁-receptor density was also increased in the dorsal striatum contralateral to the kindled amygdala of short-term rats. The short-term effects support the notion that limbic seizures can modify the lateral imbalance of DA activity in the striatum.     

Functional role of the conserved proline in helix 6 of the human bradykinin B2 receptor

Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B₂ receptor (B₂R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B₂R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B₂R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism.     

Bradykinin-induced biphasic response in the rat isolated stomach fundus: Functional evidence for a novel bradykinin receptor

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg⁹-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl₂ (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-depeendent reappearance of the relaxant responses, suggesting “de novo” synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 μM), atropine (1 μM), captopril (3 μM), prazosin (1 μM) or glibenclamide (1 μM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectivley by the B₁ receptor antagonist des-Arg⁹-[Leu ⁸]-BK (DAL- BK) (1 μM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B₂ receptors including NPC 431 (Thi^5,8, D-Phe⁷)-BK, NPC 349 (D-Arg Hyp³, Thi^5,8, D-Phe⁷)-BK, NPC 567 (D-Arg -Hyp³, D-Phe⁷)-BK and NPC 361 (D-Phe⁷)-BK (3 to 10 μM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B₁ and B₂ selective BK receptor antagonists.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor

The bradykinin (BK) B₁ receptor (B₁R) is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B₁R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP) (enhanced green FP [EGFP] or mCherry) prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg⁹-BK, [Leu⁸]des-Arg⁹-BK, respectively). The design of the spacer-ligand joint peptide was validated by a competition assay for [³H]Lys-des-Arg⁹-BK binding to the human B₁R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B₁R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy). Both assays indicated that the best design was FP-(Asn-Gly)_n-Lys-des-Arg⁹-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B₁R antagonist. EGFP-(Asn-Gly)₁₅-Lys-des-Arg⁹-BK competed for the binding of [³H]Lys-des-Arg⁹-BK to human recombinant B₁R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg⁹-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B₂ receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B₁R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology).     

Development of novel adenosine receptor ligands based on the 3-amidocoumarin scaffold

With the aim of finding new adenosine receptor (AR) ligands presenting the 3-amidocoumarin scaffold, a study focusing on the discovery of new chemical entities was carried out. The synthesized compounds 1–8 were evaluated in radioligand binding (A₁, A_2A and A₃) and adenylyl cyclase activity (A_2B) assays in order to determine their affinity for human AR subtypes. The 3-benzamide derivative 4 showed the highest affinity of the whole series and was more than 30-fold selective for the A₃ AR (K_i = 3.24 μM). The current study supported that small structural changes in this scaffold allowed modulating the affinity resulting in novel promising classes of A₁, A_2A, and/or A₃ AR ligands. We also performed docking calculations in hA_2A and hA₃ to identify the hypothetical binding mode for the most active compounds. In addition, some ADME properties were calculated in order to better understand the potential of these compounds as drug candidates.     

The role of β2-glycoprotein I (β2GPI) carbohydrate chains in the reactivity of anti-β2GPI antibodies from patients with primary antiphospholipid syndrome and in the activation and differentiation of U937 cells

Several studies have shown that conformational changes of β₂-glycoprotein I (β₂GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-β₂GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of β₂GPI in this anti-β₂GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-β₂GPI antibodies from APS patients with native, partially glycosylated β₂GPI (pdβ₂GPI; without sialic acid) and completely deglycosylated β₂GPI (cdβ₂GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-β₂GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three β₂GPI forms were also studied. We found an increased reactivity of anti-β₂GPI against pdβ₂GPI and cdβ₂GPI compared to native β₂GPI. Both deglycosylated β₂GPI isoforms showed higher inhibition of the anti-β₂GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/β₂GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in β₂GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.